Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

Abstract

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at "canonical" position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1' substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

Carboxypeptidase O is a lipid droplet-associated enzyme able to cleave both acidic and polar C-terminal amino acids

“…Most notably, the above data confirms that CPO is functional as an enzyme within the early secretory pathway, and suggests a method to examine the intracellular substrate specificity of CPO in living cells. Previous in vitro analyses of CPO substrate specificity indicated that CPO is able to cleave C-terminal glutamate [ 20 ]; cleavage of aspartate was recently demonstrated in vitro [ 21 ]. CPO is unable to cleave C-terminal basic or hydrophobic residues, with the exception of a very weak ability to cleave C-terminal alanine, and the cleavage of polar amino acids has not yet been investigated [ 20 ].…”

“…While other digestive CPs had a prodomain thought to be necessary for folding and regulation [ 18 , 19 ], CPO lacked this feature and was predicted to be an inactive carboxypeptidase homolog. It has now been shown that CPO produces a fully functional enzyme even in the absence of a prodomain, is GPI-anchored, and is expressed on the surface of intestinal enterocytes where it likely processes dietary proteins and peptides [ 20 , 21 ]. The ability of CPO to cleave C-terminal acidic amino acids suggests that CPO complements the functions of CPA and CPB in the digestion of dietary proteins [ 20 ].…”

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

“…2). CPA-like enzymes are predicted to cleave aliphatic or aromatic C-terminal amino acids due to a hydrophobic amino acid at the bottom of their speci city pocket (residue 255 in bovine CPA1), while CPBlike enzymes are predicted to cleave basic C-terminal amino acids with an acidic amino acid, often aspartic acid, at their residue 255 equivalent [18]. Fungal proteins identi ed within the CPB clade often had an Asp at position 255; however, a lot of variation was seen at this position, as nearly every amino acid was represented.…”

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

“…This ambiguity is further supported by the fact that the active site residues from these basal fungi enzymes are not at all different, all exhibiting CPA-like character. CPA-like enzymes are predicted to cleave aliphatic or aromatic C-terminal amino acids due to a hydrophobic amino acid at the bottom of their speci city pocket (residue 255 in bovine CPA1), while CPBlike enzymes are predicted to cleave basic C-terminal amino acids with an acidic amino acid, often aspartic acid, at their residue 255 equivalent [18].…”